Environmental estrogens are substances present in the environment that may mimic or antagonize the actions of naturally occurring estrogens, such as 17beta-estradiol, in humans and other animals. Although their reproductive effects have been intensively studied, what, if any, effect most environmental estrogens may have on neuronal development is largely unknown. Estradiol, however, modulates the development of many parts of the nervous system, including the cerebral cortex and hippocampus. Therefore, insofar as environmental estrogens mimic or antagonize the actions of estradiol, they would be expected to alter brain development. This proposal will investigate the effects of environmental estrogens on the development and physiology of hippocampal and cortical neurons in vitro. Because of their reported potency in binding to nuclear estrogen receptors, activating normally estrogen-stimulated gene transcription, stimulating proliferation in estrogen-dependent cell types, and activating other cellular processes that are also stimulated by estradiol, we have chosen to investigate three substances belonging to very different categories of environmental estrogens: the fungal metabolite zearalenone, the PCB 3,3',4,4'- tetrachlorobiphenyl, and the alkylphenol 4-octylphenol. The specific aims are: (1) To determine the actions of these environmental estrogens on neuronal differentiation in vitro. We will measure the proportion of neurons that develop neurites and the elongation and branching of the resulting axons and dendrites. (2) To determine their actions on intracellular calcium homeostasis, including intracellular free Ca2+ ion concentrations ([Ca2+]i), Ca2+ influx, and release of Ca2+ from intracellular stores. (3) To determine their actions on nitric oxide metabolism, including induction and activation of nitric oxide synthase in neurons, and activation of purified nitric oxide synthase in vitro. The long-term goal of these studies is to determine whether environmental estrogens alter neuronal development and to determine the cellular mechanisms of such effects.